This revised application is a continuation of R29 DC02398, "Genetic Mapping of a Syndrome with Hearing Impairment." Our research has focused on the molecular basis of craniofacial disorders, in particular Crouzon craniofacial dysostosis (CFD), which is characterized by craniosynostosis (premature closure of calvarial sutures) and mapping CFD and delineating the entire structure of the mutated gene, FGFR2. We determined that a single point mutant osteoblasts. Based upon these observations, we now hypothesize that the default pattern of tissue growth, when two like tissues come into proximity, is fusion. The normal development of the calvarium (which requires like tissues not to fuse) would then be dependent on complex genetic systems to override the natural fusion process, perhaps by increasing sutural apoptotic rates. Craniosynostosis would result from a reversion to the default (fusion) pathway of tissue growth. Thus, constitutive signaling from mutated FGF4s causes a dominant gain of function initiating involved. Newly developed nucleic acid array technologies permit the comprehensive evaluation of the entire set of expressed genes (the expressome) throughout the course of abnormal suture development. Thus, for this new proposal, we devised a chimeric nude rat model, which when implanted with mutant human osteoblasts beneath the calvarium, develops premature synostosis. The differential gene expression patterns between samples from this novel in vivo model, related in vitro cell co- cultures, and controls will be analyzed across time to identify the downstream genes involved in craniosynostosis. Laboratory techniques for analyzing samples will include differential display and robotically gridded nucleic acid arrays. Gene analysis will be prioritized based upon homology to genes known to control multicellular tissue growth, cell contact and adhesion, and osteoblasts processes active in craniosynostosis. Finally, the differentially expressed genes will be related to the biology of cranial development by 1) in situ hybridization for analysis of spatial and temporal patterns; and 2) evaluation of impact on apoptosis for assessment of physiological effect. The delineation of the panoply of genes involved in causing craniosynostosis will enhance our understanding of these diseases and suggest new therapeutic strategies.